Methods for determination of an analyte

ABSTRACT

The invention relates to analytical methods in which the partition of a labeled substance between a liquid and a solid phase is determined. The assays include solid-phase reagents which can be particulate or monolithic such as, for example, a coated tube. Assays of this type are known per se to the person skilled in the art and include immunoassays and immunometric assays.

The invention relates to analytical methods in which the partition of a labeled substance between a liquid and a solid phase is determined. The assays include solid-phase reagents which can be particulate or monolithic such as, for example, a coated tube. Assays of this type are known per se to the person skilled in the art and include immunoassays and immunometric assays.

In many cases incubation of the reagents is followed by removal of the solid phase from the liquid phase, so-called heterogeneous or solid-phase immunometric determinations. A particulate solid phase can be removed from the liquid phase by sedimentation, centrifugation, filtration or a magnetic field.

Solid phases for use in such immunoassays are likewise known to the person skilled in the art and described in the literature, see, for example, VOLLER, A. et al., Bull. World Health Org. 53, 55-65 (1976).

Magnetizable particles which can be suspended but are insoluble in the liquid phase have the advantage that they can rapidly be removed from the liquid phase in a magnetic field.

Methods for the determination of analytes using magnetic particles are described, for example, in U.S. Pat. Nos. 4,554,088, 3,933,997 and in European Patent Application EP 0 136 126. Substances labeled with radioisotopes are employed for determination of an analyte in these methods.

These methods described above have the crucial disadvantage that they are confined to the use of radioisotopes with their known potential hazards and the consequent difficulties with handling and waste disposal.

Now widely used as alternative to the use of radioisotopes as label are enzymes and thus enzyme immunoassays. In this method the reagent to be labeled is linked to an enzyme and, after the formation of the solid-phase receptor/analyte complex or of the solid-phase receptor/analyte/receptor complex, an incubation is carried out with a substrate of the enzyme. The enzyme reaction now produces a colored, luminescent or fluorescent derivative of the substrate. After completion of the reaction it is the absorption, luminescence or fluorescence which is measured in place of the radioactivity.

Suitable particles which can be removed magnetically have an intrinsic color owing to their content of, for example, iron oxide particles. They absorb light as a function of their size and their magnetite content. This intrinsic absorption by the particles reduces non-specifically the signal in a measurement of absorption, luminescence or fluorescence. The extent of the absorption of light depends, inter alia, on the amount of magnetic particles employed.

The dependencies also apply, of course, although the extent differs, to particles not amenable to magnetic attraction.

The Patent Application EP 0 177 191 describes a heterogeneous enzyme immunoassay with magnetic particles as solid phase, with an enzyme/analyte conjugate and with a chromogenic substrate as signal-emitting reagent. The magnetic particles must be removed before measuring the absorption. The disadvantage of this method is the necessity for an additional further incubation step of, normally, 20-60 min to generate the signal. The method thus requires a considerably greater expenditure of time and effort than comparable methods which use radioisotopes. Another disadvantage is based on the fact that this method cannot be used for heterogeneous immunoassays which use a direct chemiluminescent or fluorescent label.

This is not possible in enzyme-enhanced luminescence systems as are disclosed in the literature and employed, for example, in the Patent Application EP 0 149 565. A peroxidase is used therein to stimulate a substrate of the luminol type to emit light. Removal of the particles which carry the complex of receptor and enzyme in this method means that chemiluminescence generation is no longer possible and thus no signal can be measured either.

U.S. Pat. No. 4,576,912 discloses a method in which a fluorophore is coupled via a carrier molecule to the specific binding partner. The fluorophore is liberated after removal of the unbound conjugate by chemical or enzymatic hydrolysis of the carrier molecule, and this hydrolysis typically takes 60 min. Chemiluminescent labels are not considered in the patent.

The method described in EP 0 149 565 represents the state of the art. This patent application describes the measurement of chemiluminescence and fluorescence in the presence of suspended magnetic particles. It is shown therein that there is a narrow concentration range for the magnetic particles within which it is possible to carry out an assay despite the absorption of light (see also FIG. 1). However, as the present invention shows, this does not exhaust the advantages of the magnetic particle technology such as large surface area and short diffusion pathways.

It has now been found, surprisingly, that it is possible by adding suitable substances to measure without interference the signal of the labeled reagents by detaching the label and after removal of the solid phase. The measured signal shows a strict relation to the concentration of the analyte employed.

The method according to the invention achieves a distinctly greater signal yield and improved signal dynamics than the method described in EP 0 149 565 (see also FIG. 2).

Without wishing to specify a particular mechanism of action hereby, we assume that the substances according to the invention split the label-carrying complex with the solid phase and liberate the label in a suitable form.

The present invention now makes it possible to employ the solid phase in high concentrations. The enlarged surface area also makes it possible to employ the solid-phase receptor in a larger excess compared with the analyte. Furthermore, the diffusion pathways are considerably shortened. The two effects have a beneficial influence on the position of the equilibrium and on the reaction kinetics of an assay.

The present invention now relates to a heterogeneous immunochemical method for the determination of an analyte in a liquid medium, which contains the following steps in the method:

-   -   incubation of a sample which contains the analyte with the         magnetically attractable solid-phase reagent and with the         reagent labeled with a luminophore or fluorophore in a reaction         vessel, the labeled reagent being partitioned between liquid         phase and the magnetically attractable particles as a function         of the concentration of the analyte,     -   removal of the liquid phase from the solid-phase particles,     -   resuspension of the particles in a reagent A, removal of the         particles,     -   elicitation and measurement of the luminescence or fluorescence         reaction in the supernatant,         where reagent A is suitable for detaching the label from the         solid phase.

It is possible, where appropriate, for washing steps to be inserted between the individual steps.

The process is, in principle, also suitable for systems which, for example, determine enzyme activities or radioactivity as marker, however it is particularly advantageous in connection with a fluorescent or chemiluminescent label, and a chemiluminescent label is preferred.

Various chemiluminescence systems are described in the literature, and of these luminol, isoluminol and acridinium derivatives have acquired major importance. Luminol and isoluminol derivatives are described by H. R. Schroeder et al. “Methods in Enzymology” Academic Press Inc, New York, Vol LVII 424 et seq. The use of the acridinium derivatives as labeling substances is described by Weeks et al. (Clin. Chem 29/8, 1983, 1474-1479). The chemiluminescence of acridinium derivatives can be initiated by addition of alkaline H₂O₂ and measured within a few seconds.

These compounds have the advantage as labeling reagent that no further incubation step is necessary to elicit the signal, in contrast to the enzyme-enhanced luminescence systems. Furthermore, the acridinium compounds are stable to the reagents used herein.

Preferred chemiluminescent labels are the acridinium compounds described in EP 0 257 541 and EP 0 330 050.

The exact construction of the magnetically attractable particles used herein is known per se to the person skilled in the art and substantially unimportant in respect of the invention, preference being given to superparamagnetic particles which can advantageously be designed as composite particles or pure magnetite particles. It is also possible in principle for non-magnetizable particles to be employed, in which case the separation method must be appropriate for the particular particle, for example centrifugation or chromatography. Such particulate solid phases are also known to the person skilled in the art, such as, for example, latex particles which can be colored or uncolored.

The particles preferably have a size of 0.1-5.0 μm, and a range of 0.2-2.5 μm is particularly preferred, very particularly preferably 0.2-1.0 μm.

It is advantageous to employ in an assay small particles which, for the same total mass of particles, have a large number and a large surface area overall. A large number of particles means that the distance from particle to particle in a given volume is very small, the consequence being that the diffusion pathways of the dissolved analytes and of the labeled substance to the particle surface area are very small. There is accordingly an increase in the rate of the reaction leading to the formation of the receptor complexes bound to the solid phases. Moreover, the larger total surface area of the solid phase makes it possible to achieve an increased receptor concentration. High receptor concentrations result in extensive binding of the analyte and likewise in an increase in the reaction rate.

Small magnetically removable composite particles do, however, require a large proportion of magnetizable material in order to be able to be removed from the reaction solution within practicable times. The absorption of light by a particle suspension depends, inter alia, on the nature, on the size, on the content of magnetic material and on the concentration of the particles employed. If, as described in the state of the art, the signal is measured in the presence of the suspended particles, the measurable signal in an assay for the determination of, for example, TSH in serum then passes through a maximum as the amount of particles per mixture increases. When the amount of particles which is optimal under these conditions is exceeded, the increase in the absorbed amount of light exceeds the additional chemiluminescence obtained by the increase in the binding of labeled reagent (FIG. 1).

The use of magnetically removable particles in a sufficiently wide concentration range is therefore made possible only by the method according to the invention.

The detachment reagents used in this invention meet the following requirements:

-   -   elimination of at least one intermolecular linkage which is         essentially composed of hydrogen bonds and ionic, hydrophobic         and van der Waals interactions     -   no impairment of the chemiluminescence reaction by absorption of         light     -   no quenching of the excited state of the luminophore     -   no reaction with alkaline H₂O₂ solution     -   no impairment of the magnetic removability of the magnetic         particles     -   no dissolution or aggregation of the magnetic particles.

Reagents which meet these conditions include, for example, chaotropic reagents, ionic detergents, some organic solvents, and buffer substances which are suitable for adjusting a pH in the range 1.5-3. Besides the reagents listed hereinafter by way of example, a large number of other reagents and their combinations which meet the stated requirements are conceivable. Some of them have been employed in the examples. Particularly Preferred preferred Concentration range chaotropic reagents: KSCN 2 mol/l-sat.*⁾ 3-5 mol/l Guanidinium hydrochloride 3 mol/l-sat. 3-5 mol/l very particularly preferred 3 mol/l, pH 4 CaCl₂ 1 mol/l-sat. 1-3 mol/l NaClO₄ 1 mol/l-sat. 1-3 mol/l Tetra-N-butylammonium 0.01% (w/v)- 0.01-1% chloride sat. (w/v) Tetramethylammonium 0.01% (w/v)- 0.01-1% chloride sat. (w/v) Urea 3-8 mol/l 4-7 mol/l very particularly preferred 5 mol/l, pH 4 ionic detergents: SDS (Na dodecyl sulfate) 0.01-20% 0.01-10% (w/v) (w/v)-sat. very particularly preferred 1% (w/v) in 10 mmol/l sodium phosphate, pH 6 organic solvents: Propionic acid 0.1-30% (w/v) 1-10% (w/v) Ethylene glycol 30-80% (w/v) 40-60% (w/v) DMF (dimethylformamide) 40-90% (v/v) 50-70% (v/v) very particularly preferred 60% (v/v) buffer: Glycine pH 2 0.1 mol/l-sat. 0.5-1 mol/l very particularly preferred 0.5 mol/l, pH 2 *⁾sat. = saturated solution

These reagents can be employed singly or in combination.

It is in principle easy for the person skilled in the art to establish the optimal conditions for a particular substance or combination of substances by a few experiments in which he employs various concentrations.

The embodiments indicated in the examples are particularly preferred.

The invention can in principle be used in methods in which the partition of a labeled reagent between a liquid and a solid phase is determined.

In the course of this method, the solid phase, analyte and labeled reagent are incubated, either simultaneously or consecutively, for a suitable time. After the incubation the solid phase is removed and then suspended in the detachment reagent and incubated for a short time. The particles are again removed and the supernatant is measured. If a large number of samples is investigated at the same time, the sequence of steps is typically as follows:

-   A. Pipetting of suspended, magnetically attractable particles,     samples, standards and labeled reagent into reaction vessels. -   B. Incubation of the vessels containing the reaction mixture at the     required temperature for a defined time. -   C. Removal of the magnetic particles with an arrangement of magnets     suitable for the vessels used. -   D. Removal of the supernatants by aspiration with suitable pipettes,     pumps or vacuum. -   E. Repeated addition and removal by aspiration of washing buffer. -   F. Removal or switching off of the magnetic field used to remove the     magnetizable particles, and resuspension of the magnetic particles     in the detachment reagent. -   G. Removal of the particles with the magnet system, transfer of the     supernatant into a suitable vessel and measurement of the label.

The claims form part of the disclosure.

The following examples serve to illustrate the invention but do not restrict it in any way. Unless otherwise indicated, Estapor® particles (0.35 μm) supplied by Rhone-Poulenc are used as magnetically removable particles.

EXAMPLE 1

Determination of Human Thyroid Stimulating Hormone (hTSH) According to the State of the Art (Measurement in Suspension)

The following reagents were incubated together in polystyrene tubes:

-   -   0.006 mg; 0.013 mg; 0.019 mg; 0.025 mg; 0.050 mg; 0.075 mg; 0.1         mg and 0.125 mg of magnetically removable particles coated with         a monoclonal anti-hTSH antibody,     -   100 μl of a solution 50 μIU/ml hTSH in human serum,     -   40 μl of a solution of a monoclonal antibody conjugated to an         acridinium derivative,     -   suspension of the particles,     -   incubation of the suspension at 37° C. for 15 min,     -   removal of the particles with a separation system supplied by         Gene-Trak, and removal of the supernatant by aspiration,     -   300 μl of washing buffer are added four times, and removed by         aspiration again each time, to the removed particles,     -   the particles are then resuspended in 100 μl of washing buffer,     -   and transferred into polypropylene tubes (1 ml). These tubes are         placed in polystyrene tubes (5 ml). The chemiluminescence is         elicited by addition of 300 μl of 0.1 N nitric acid and 0.5%         H₂O₂ and 300 μl of 0.25 N sodium hydroxide solution and measured         in a luminescence measuring apparatus supplied by Berthold         (Model LB952T) (see FIG. 1).

EXAMPLE 2

Determination of Human Thyroid Stimulation Hormone (hTSH)

The following reagents were incubated together in polystyrene tubes:

-   -   0.025 mg; 0.125 mg and 0.250 mg of magnetically removable         particles coated with a monoclonal anti-hTSH antibody,     -   100 μl of a solution 50 μIU/ml hTSH in human serum,     -   40 μl of a solution of a monoclonal antibody conjugated to an         acridinium derivative,     -   suspension of the particles,     -   incubation of the suspension at 37° C. for 15 min,     -   removal of the particles with a separation system supplied by         Gene-Trak, and removal of the supernatant by aspiration,     -   300 μl of washing buffer are added four times, and removed by         aspiration again each time, to the removed particles,     -   the particles are then resuspended in 100 μl of removal reagent,         and then         -   a) the suspension is employed for measurement         -   b) the detached magnetic particles are removed and the             supernatant is employed for measurement         -   c) the removed magnetic particles from (b) are resuspended             in 100 μl of washing buffer and this suspension is employed             for measurement,

the sample employed for measurement is transferred into polypropylene tubes (1 ml). The tubes are placed in polystyrene tubes (5 ml). The chemiluminescence is elicited by addition of 300 μl of 0.1 N nitric acid and 0.5% H₂O₂ and 300 μl of 0.25 N sodium hydroxide solution and measured. TABLE 1 Removal of the magnetic particles before signal detection [hTSH assay (signal in RLU)] Supernatant after Removed magnetic Measurement in detachment and particles suspension removal (resuspended) 1) GuaHCl 0.025 mg 349,921 236,317 223,615 0.125 mg 188,512 353,445 37,410 0.250 mg 132,766 295,127 10,692 2) Urea 0.025 mg 240,053 286,195 49,715 0.125 mg 158,493 305,402 5,961 0.250 mg 117,389 270,993 3,778 3) SDS 0.025 mg 282,840 331,649 3,198 0.125 mg 240,143 395,635 5,817 0.250 mg 156,367 398,028 6,946 4) DMF 0.025 mg 171,610 139,728 64,795 0.125 mg 129,485 135,785 38,591 0.250 mg 108,097 185,875 20,312 Detachment reagent: 1) Guanidinium HCl 3 mol/l, pH 4 2) Urea 5 mol/l, pH 4 3) SDS 1 g/100 ml of 10 mmol/l sodium phosphate buffer pH 6 4) DMF 60% (w/v) These data are depicted as a graph in FIG. 2.

EXAMPLE 3

Determination of Human Thyroid Stimulating Hormone (hTSH)

The following reagents were incubated together in Flow tubes:

-   -   0.125 mg of magnetically removable particles coated with a         monoclonal anti-hTSH antibody,     -   100 μl of a standard solution of hTSH in human serum,     -   40 μl of a solution of a monoclonal antibody conjugated to an         acridinium derivative,     -   suspension of the particles,     -   incubation of the suspension at 37° C. for 15 min,     -   removal of the particles with a separation system supplied by         Gene-Trak, and removal of the supernatant by aspiration,     -   300 μl of washing buffer were added four times to the removed         particles,     -   the particles were then resuspended in 100 μl of removal reagent         and subsequently placed in the separation system for 2 min,

the supernatant is removed and transferred into Flow tubes. The Flow tubes are placed in Sarstedt tubes. The chemiluminescence is elicited by addition of 300 μl of 0.1 N nitric acid and 0.5% H₂O₂ and 300 μl of 0.25 N sodium hydroxide solution and measured luminescence measuring apparatus. TABLE 2 No removal Removal of the of the particles particles Standard RLU RLU S0 0.0 μIU/ml 273 445 S1 0.14 μIU/ml 1219 1149 S2 0.47 μIU/ml 3179 2060 S3 1.5 μIU/ml 9235 5621 S4 4.8 μIU/ml 31477 17078 S5 14.2 μIU/ml 96629 49972 S6 50 μIU/ml 304099 164966 S7 100 μIU/ml 496261 276073

These data are depicted as a graph in FIG. 3. 

1. A heterogeneous immunochemical method for determination of an analyte in a liquid medium, which contains the following steps in the method: incubation of a sample which contains the analyte with the particulate, attractable solid-phase reagent and with the reagent labeled in a reaction vessel, the labeled reagent being partitioned between liquid phase and the solid-phase reagent as a function of the concentration of the analyte, removal of the liquid phase from the solid-phase particles, resuspension of the particles in a reagent A, removal of the particles, elicitation and measurement of the luminescence or fluorescence reaction, wherein reagent A is suitable for detaching the label from the solid phase.
 2. The method as claimed in claim 1, wherein the particulate solid phase is magnetically attractable.
 3. The method as claimed in claim 1, wherein reagent A contains in an aqueos solution one or a combination of at least two substances of the following classes of reagents: chaotropic reagents ionic detergents organic solvents buffers in the pH range 1.5-3.
 4. The method as claimed in claim 1, wherein reagent A contains in an aqeous solution one or a combination of at least two substances of the following class of reagents: KSCN guanidinium hydrochloride CaCl₂ NaClO₄ tetra-N-butyl-ammonium chloride tetramethylammonium chloride urea SDS propionic acid ethylene glycol DMF glycine in the pH range 1.5-5. 